~———— ——— . . . ' v . . ‘ [- ... ... . ‘.v . - v ‘ .. . ~ x- ' ‘ v' -" - " " , -. - - - ‘ ‘ THE SURVIVAL OF ESCHERICHlA COLL MICROCOCCUS FLAVUS AND BACILLUS SUBTILES DURWG SPRAY DRYING 0F SKEMMILK AND STGRAGE OF SKEMMILK POWDER Thesis for the Degree 6! M. s. MICHIGAN STATE umvmsm STERLING. SAMUEL THOMPSON 1975 NF gag C ‘ ABSTRACT THE SURVIVAL OF ESCHERICHIA COLI, MICROCOCCUS FLAVUS AND BACILLUS SUBTILIS DURING SPRAY DRYING OF SKIMMILK AND STORAGE OF SKIMMILK POWDER BY Sterling Samuel Thompson Investigations were conducted to determine the survival of Escherichia coli, Bacillus subtilis and Micrococcus flavus inoculated into concentrated skimmilk which was spray dried under varying operating conditions. On separate occasions 50 gallons of raw whole milk were separated into skimmilk and cream fractions. The skim- milk fraction was pasteurized at 145 F (62.8 C) for 30 minutes and concentrated to 35-40% total solids in a pilot plant vacuum pan. The approximate ratio of concentration was 4.3:1. A hydrogen peroxide—catalase treatment was used as an adjunct to the pasteurization process. The concentrated skimmilk was heated to 120 F (48.9 C) and sufficient H202 to give a 0.05% concentration was added to the sample for a contact time of 20-30 minutes. The sample was then cooled to 100 F (37.7 C) and an appropriate amount of sterile catalase was added to decompose the Sterling S. Thompson the H202 to water and oxygen. Standard plate counts were performed on each sample to determine the bactericidal efficiency of the treatment. The plate counts indicated that H202 was a beneficial adjunct to pasteurization. Concentrated milk was inoculated with a pure broth culture concentration of l x 106 organisms/ml of Escherichia coli, Bacillus subtilis or Micrococcus flavus and spray dried at three different exit air temperatures, 160, 180 and 200 F (71.1, 82.2, and 93.3 C). While spray drying at various exit air temperatures reduced the numbers of each organism, in no case did it yield bacterial- Ifree powder with the amount of inoculum used. Under all operating conditions E. subtilis was much more resistant to spray drying, followed by E. flavus, and E. 99;; demon- strated the least resistance. The skimmilk powders were stored at 25 C (77 F) and evaluated for progressive microbial changes in the product. While substantial reductions were observed with E. 92;; over 1 to 6 months storage, E. subtilis and E. flavus reductions were less over similar periods of storage. Spray drying at high temperatures resulted in powders with low moisture contents. This combination of heat and low moisture influenced the survival of E. EQEE, E. subtilis and E. flavus during drying and storage, how- ever, these factors do not provide absolute microbial control. THE SURVIVAL OF ESCHERICHIA COLI, MICROCOCCUS FLAVUS AND BACILLUS SUBTILIS DURING SPRAY DRYING OF SKIMMILK AND STORAGE OF SKIMMILK POWDER BY Sterling Samuel Thompson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1975 ACKNOWLEDGMENTS The author wishes to express his appreciation to Dr. L. G. Harmon his major professor for his guidance and assistance during the course of this study and in prepara— tion of this manuscript. Thanks are also extended to Dr. C. M. Stine of the Department of Food Science and Human Nutrition and Dr. J. W. Allen of the Department of Marketing and Transpor- tation Administration for their suggestions as members of the guidance committee. Special thanks to Marguerite Dynnik for her assistance in the laboratory. Appreciation also is extended to Clem Kuehler and Bruce Harte for their assis- tance with the operation of the spray drier throughout this research. Finally, I wish to thank my wife, Barbara, and Lovaleria King for their help in editing the text and pre- paring the final manuscript. ii LIST OF TABLES . . . LIST OF FIGURES . . INTRODUCTION . . . . LITERATURE REVIEW . TABLE Microorganisms in Milk OF CONTENTS Powder Microbiological Standards for Dried Milk Powder . . . . Types of Microorganisms in Milk Powder . . Changes in Microbial Content During Storage Significance of Microbial Counts on Milk Powder . . . . Control of the Microbial Content of Dried Milk . . . . . Effect of Spray Drying on Various Micro- organisms . . MATERIALS AND METHODS Selection and Propagation of Microorganisms Preparation of Concentrated Skimmilk . . . Hydrogen Peroxide-Catalase Treatment Spray Drying . . Moisture Determinations Microbiological Analyses of Skimmilk iii Powder Page vi 10 17 21 24 26 29 29 3O 31 34 35 38 RESULTS . . . . . . . . . . . . . . . . . . . . Microbiological Analyses of Concentrated Skimmilk . . . . . . . . . . . . . . . . . Effect of Spray Drying Conditions on Initial Microbial Populations in Skimmilk Powder . Effect of Storage on Microbial Populations of Spray Dried Skimmilk . . . . . . . . . Effect of Spray Drying Temperature on the Moisture Content of Skimmilk Powder . . . DISCUSSION 0 O O O O C O O O O O O O O O O O O 0 Effect of Spray Drying on Microbial Content of Skimmilk Powder . . . . . . . . . . . . Effect of Storage and Moisture Content on Survival of Microorganisms in Spray Dried Skimmilk . . . . . . . . . . . . . . Effect of Spray Drying on the Moisture Content of Skimmilk Powder . . . . . . . . SUMMARY AND CONCLUSIONS . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . . . GENERAL REFERENCES 0 O O O O O O O O O O C O O 0 iv Page 40 40 40 43 48 50 50 51 54 56 59 65 LIST OF TABLES Specific bacterial grading requirements for dried milk . . . . . . . . . . . . . . . . Specific moisture standards for milk powders . Drying conditions used to prepare skimmilk powder . . . . . . . . . . . . . . . . . . . . Plate counts of concentrated milk after treatment with H202 and catalase . . . . . . . Effect of various temperatures used in spray drying skimmilk on destruction of E. coli, E. subtilis and E. flavus when milk contained 1 x 10 organisms/ml . . . . . . . . . . . . . Effect of storage on the number of E. coli in spray dried skimmilk powder . . . . . . . . Effect of storage on the number of E. flavus in spray dried skimmilk powder . . . . Effect of storage on the number of E. subtilis in spray dried skimmilk powder . . . Moisture content of spray dried skimmilks . . Page 19 36 41 42 47 47 48 49 Figure 1. LIST OF FIGURES Page Survival of E. coli in spray dried skim— milk during Etorage at 25 C in milk dried with exit air temperatures of 160, 180 and 200 F . . . . . . . . . . . . . . . . . . . 44 Survival of M. flavus in spray dried skim- milk during gtorage at 25 C in milk dried with exit air temperatures of 160, 180 and 200 F . . . . . . . . . . . . . . . . . . . 45 Survival of B. subtilis in spray dried skim- milk during Etorage at 25 C in milk dried with exit air temperatures of 160, 180 and 200 F . . . . . . . . . . . . . . . . . . . 46 vi INTRODUCTION The U.S. Department of Agriculture reported that in 1972, 1,223,456 pounds of nonfat dry milk were manu— factured in the United States (55). Nonfat dry milk is used in the preparation of many food products including bread, sausage, ice cream and cottage cheese. Because of its varied usage, it is essential that NFDM be wholesome and free from objectionable bacteria. The bacterial con- tent of NFDM is of concern quantitatively and with respect to the types of microorganisms. Recognition of a rela- tionship between microbial counts on dairy products, such as NFDM, and the care used in production and handling resulted in the establishment of microbiological standards by Federal and state agencies. The microbial content of any food product is usually an index of conditions under which the product was produced and handled, and of the keeping quality. In general NFDM and other dry milk products are prepared almost exclusively by spray drying. Spray drying at high temperatures reduces the microbial population, but does not render the dried product completely free of microorganisms. Much of the recent research conducted on microbial populations of spray dried NFDM has been con- centrated on the survival of certain pathogenic organisms, Salmonella and Staphylococcus aureus. The literature con- tails little about the survival of some of the typical contaminants or spoilage type microorganisms. The objective of this investigation was to deter— mine the effects of various spray drying conditions used in producing milk powder on the viability of Bacillus subtilis, Micrococcus flavus and Escherichia coli. E. subtilis is a typical aerobic spore forming microorganism which causes spoilage in some dairy products. Hammer and Babel (23) reported that this organism caused coagulation of evaporated milk. Since E. subtilis is a thermoduric microorganism, survival of large numbers of this organism under inadequate heating conditions may cause defects, particularly in reconstituted milks. Breed et a1. (6) indicated that E. flavus is fre- quently found in milk, other dairy products and on dairy utensils. E. flavus survives conventional pasteurization, and inadequate thermal processing or improper sanitation may cause contamination problems. Neither E. subtilis nor E. flavus is significant from the standpoint of public health. However, substantial increases in the number of these organisms in NFDM will cause an undesirable increase in total plate count. A higher than acceptable total bacterial count will cause the failure of NFDM to meet specific grading requirements established by the American Dry Milk Institute. Traditionally strains of E. 92;; have been con- sidered indicators of fecal contamination and their presence in dairy products suggests unsanitary conditions or prac- tices during production, processing and/or storage. Hall and Hauser (20) and Insalata (31) suggested that certain serotypes of E. ggll_may produce food-borne disease. According to Jay (33) enteropathogenic E. coli strains differ from the more normal E. 321$ strains by being more virulent and by reacting with E. 99;; OB and O antisera. Occurrence of outbreaks of gastroenteritis associ- ated with certain E. 99;; serotypes makes it essential that the food industry become more aware of the potential prob- lems this organism may cause if proper sanitary conditions are not maintained. Two important factors associated with influencing the microbial content of NFDM are temperatures used prior to drying and method of drying. Frazier (18) indicated that prior to spray drying, milk should be concentrated two or three times and preheated to a temperature ranging from 145 F to 200 F. This preliminary heat treatment would pasteurize the milk, thereby inactivating the less heat stable microorganisms. Other factors which can also influence microbial content are the extent of contamination of the original raw milk, pasteurized milk, processing equipment, and introduction of contaminants during packaging. REVIEW OF LITERATURE In recent years spray dried milk products con- taminated with E. aureus enterotoxin have caused several food poisoning outbreaks (2, 4). In addition, contami- nation of spray dried milk with Salmonella has generated several investigations including those by McDonough and Hargrove (42), Marth (44), LiCari and Potter (38, 39). On the other hand the literature contains little infor- mation concerning survival and growth characteristics of contaminants and/or spoilage type microorganisms in spray dried powders during manufacture and storage under either adverse or normal conditions. Therefore, this review will present a general survey of pertinent factors involving the presence of both non-pathogenic and pathogenic micro- organisms which have been found in milk powders. Microorganisms in Milk Powder According to Jay (33) the microbial content of powdered milk may reach the range of log 6 to 8 per gram. The presence of such high numbers of microorganisms in milk powder would be attributed to the fact that the micro- organisms were concentrated on a per gram basis with the milk solids when the water was removed. 5 Frazier (18) reported that the microbial content of powdered milk depended upon the microbial content of the liquid milk to be dried, the time and temperature of pre- heating, the evaporation process, contamination and growth in storage equipment and the method of drying. During spray drying a fine mist of concentrated milk under pressure is introduced into a very hot chamber. In general, drying occurs instantaneously at the high drying temperatures. Yet, according to Crossley (10) the fluid milk does not reach sufficient temperatures during drying to insure complete destruction of all non-pathogenic or pathogenic microorganisms. Some of the initial studies involving the survival of certain non-pathogenic bacteria during drying, post processing contamination and survival of these micro- organisms during storage of dry milk powder were reported by Delepine (1914, cited by Supplee and Ashbaugh, 1922). The investigations indicated that under normal operating conditions the number of microorganisms was 10,000 to 15,000 per gram of powder, as it left the drying chamber. However, recontamination due to subsequent handling caused a sharp increase in the microbial population of the dried powder. Macy (43) reported that the temperatures employed for commercial spray drying were not sufficient to render a microbial free powder. Macy (43) also reported that a larger number of microorganisms survived during the spray drying process than during the roller drying process. The higher roller drying temperatures were effective in lowering the bacterial content of the powder. However, these temperatures were less desirable due to the degree of heat damage suffered by the finished product. Supplee and Ashbaugh (51) observed that regardless of the number of bacteria present in the liquid milk, the number which survived drying was low and did not reflect a direct relation to the number of bacteria in the liquid milk prior to drying, if the milk contained the normal flora. Their investigation demonstrated that any increase in microbial content was largely due to recontamination subsequent to drying. Crossley and Johnson (12), during their investigations of the microbial content of milk powder from two separate processing plants, also observed a lack of relationship between total counts on raw milk and powdered milk. However, they concluded that the micro- biological quality of the powdered milk depended ultimately upon the numbers and species of organisms which survived pasteurization. The drying temperatures employed caused some bacterial destruction. These early investigations and later investigations by Higginbottom (24, 27, 28), Crossley (9), Findlay et al. (16) and Olson and Nielson (47) established precedence for some of the more recent research regarding the survival of non-pathogenic as well as pathogenic microorganisms during the manufacture and storage of spray dried milks. Microbiological Standards for Dried Milk Powder Microbial standards for grades of nonfat dry milk and powdered whole milk were established by the American Dry Milk Institute and published in Standards for Grades of Dry Milks (1). According to Ingram (30) bacterial standards serve three functions: (a) minimize the risk from pathogenic organisms, (b) insure that the product was not grossly contaminated, and (c) give an estimate of product shelf-life during storage. Davis (14) reiterated the desirability of microbial standards by enumerating the following advantages: a. insure a wholesome safe product for human con- sumption. b. insure adequate keeping quality. c. indicate points of faulty processing during operation. d. improve the quality of the product. e. educate workers in hygiene and other aspects of their work. Data in Table 1 lists the bacterial standards for some dried milks. .ucmwmum ma canonm saxou Hams» no mammosumm oz .Emum “mm ca ommoxm uo: oasosm uw .xHHE mum ummcoc unnumcfl MOM umwoxm can Baum umm om omooxm uoc oasonm muosooum xHHE mac :0 unsoo Euowwaou m>HumEsmmum one "muoz m\ooo.om zamz ucmumcH mxooo.ooa numocmum m\ooo.om muuxm xasm mxooo.OOH oumncmum m\ooo.om muuxm xasm m\ooo.om muuxm m\ooo.om s5HEmum Umxomm mmw xHHz macs: who m\ooo.ooa m\ooo.ooa oumpcmum m\ooo.om o\ooo.om muuxm xaflz mun ummcoz case umpmmuo uoz cane umpmmuw uoz umaaom owumnmmOEum xmumm Emma mom mumEHumm Hmwumuomm um©3om mo momum paw mama .AHV xHHE omaup How mucmsmufisqmn mcwomum Hmwuwpomn oamwommmll.a magma 10 Types of Microorganisms in Milk Powder Higginbottom (27) suggested that due to the increased production and use of dried milk, researchers should not only be concerned with the number of bacteria but also with the type of bacteria in milk powder, espe- cially those which grow readily after reconstitution. There existed the possibility that the reconstituted milk might be held for some extended time prior to use and the inference that certain milk-containing foods were respon- sible for food poisoning made it essential that the types of organisms which survived the drying process be known. In a review of literature prior to 1949 dealing specifically with the relationship between production procedures and microbial population of milk powder, Crossley and Mattick (l3) concluded that the microflora of spray powder was directly related to the preheating temperature of the initial liquid milk, the concentrated milk, equipment and plant cleanliness. Foster et a1. (17) reported that the use of high preheating temperatures was instrumental in reducing the number of thermoduric microorganisms in the final powder. Streptococci and aerobic spore formers predominated. Pre— heating at lower temperatures resulted in the survival of large numbers of micrococci and microbacteria. The data indicated that no pathogenic organisms survived processing. Later this evidence was proven to be incorrect by the 11 research of McDonough and Hargrove (42), LiCari and Potter (38), Anderson and Stone (2) and Armijo et a1. (4) in relation to the presence of Salmonellae and E. aureus in nonfat dry milk. Observations by Cihova and Saxl (8) demonstrated that although the number of organisms isolated from dried powder processed at three separate plants differed, the types of organisms were in fact similar. B. subtilis, E. licheniformis, E. pumilus, E. cereus, E. megatherium and E. alvei occurred more frequently among the sporulating flora isolated, whereas gram positive cocci, particularly E. faecium were the predominating organisms of the total powder flora. Keogh (37) theorized that the degree or level of lactic acid or lactate in milk powder was a rough indi- cation of the number of lactic acid producing organisms in milk. (Lactic acid is thermally stable at the various heat treatments used.) In previous studies it was observed that the heat treatment the milk received during the pro- cessing of NFDM was not sufficient to eliminate all bacteria. However, unlike past assumptions that the pres- ence of the less heat resistant bacteria in milk powder was due to post processing contamination, it was suggested that the heat sensitive organisms were in the powder because the protein in the milk protected these organisms from the heat and that actual temperature of the particles 12 was less than indicated in the drying chamber. Bacterio- logical analysis of the powder demonstrated that the flora consisted mainly of spore formers and other thermoduric types such as micrococci and microbacteria. Some patho- genic organisms were also isolated including E. aureus, E. pyogenes and E. perfringens and their presence is espe- cially undesirable if the powder is incorporated as an additive to other foods. _ At the Sixth International Symposium of Food Microbiology, Planine and Milohnoja (48) presented data on one of the more recent investigations concerning the microbial content of milk powder. By analysis of variance they illustrated that daily powder samples differed in bacteriological contamination, which was attributed to the varied degrees of microbial contamination of the raw milk. The organisms which were isolated included fecal strepto- cocci, coliforms, sulphite producing clostridia, E. aureus, thermophilic and other thermoduric organisms, psychrophilic, acidophilic, caseolytic and lipolytic bacteria. A few yeasts and molds were isolated, however, no Salmonellae were detected. Galesloot and Stadhouders (19), at the same sym- posium, presented a related paper in which it was theorized that the presence of certain types of bacteria in dried milk products originated from three primary sources. 13 Raw milk. The heat treatment that the raw milk was subjected to during processing was not suf- ficient to kill all of the bacteria present. Only the thermoduric organisms survived pasteurization; Microbacterium lacticum was the most heat stable. Growth 9: organisms during the process. The entire process was conducive to bacterial growth. Growth during the process was observed with mesophilic and thermophilic microorganisms. Group D streptococci made up the major portion of the mesophilic bacteria with E. durans the dominant species. E, stearothermophilus var. calidolactis was the dominant thermophilic organism. On a few occasions E. aureus was observed. Incidental contamination. Contamination with microorganisms which did not grow during the pro- cess and caused low level contamination. The major causes of incidental contamination were related to direct human contact, air borne con- tamination during drying, cooling, transporting, instantizing and packaging. The Enterobacteriaceae (Coliform bacteria, Salmonellae) and E. aureus comprised the organisms responsible for that type of contamination. These organisms which origi— nated from incidental contamination were not related to plant conditions at the time of l4 processing but rather to the types in the pro- cessing plant. Taha et al. (52) in Part I of a dual investigation studied the microbiological quality of spray dried milk obtained from a specific processing plant. A total bacte- rial count using two sets of plates with one set incubated at 30 C (86 F) and the second set at 37 C (98.6 F) showed average counts of 13 x 106/gram and 6.8 x 106/gram, respectively. Thermoduric plates yielded an average count of 1.6 x 106/gram. This high count was attributed to the presence of large numbers of heat resistant organisms in the raw milk and to contamination during processing. Psychrophilic counts averaged 4.5 x lOS/gram. Heat treat- ment destroyed these organisms during the process, thus, their presence in the powder was due to post processing contamination. Non-pathogenic staphylococcal counts aver- aged 4 x 105/gram. Since these organisms were quite heat labile their presence was attributed to post processing contamination. Coliforms showed an average count of 6.4 x lOZ/grams and were isolated from 20% of the samples. Since they too were found to exist exclusively as non-heat resistant strains, their presence was construed to be due to post processing contamination also. Group D strepto— cocci were divided into two groups. Group I included E. faecium, E. durans and E. EQXEE, and showed an average count of 5.3 x lOS/gram. Group II consisted of E. 15 faecalis, E. gymogenes and E. liquefaciens, with an aver- age count of 9.3 x 104/gram. The presence of both groups of streptococci was attributed to their relatively high resistance to heating and drying and to recontamination following pasteurization and drying. Saccharolytic anaerobes were found in 40% of the samples, no exact number was specified. In the second half of the investigation Naguib et al. (46) identified more of the predominating micro- organisms in the spray dried milk samples. The organisms isolated, in decreasing order, were streptococci, micro- cocci, microbacteria and sporeformers. Five hundred ninety-eight cultures were isolated from plates which were incubated at 30 C (86 F) and 37 C (98.6 F). Organisms iso- lated from plates incubated at 30 C were 67.2% strepto- cocci, l9.6% micrococci, 7.4% microbacteria, 3.4% spore forming bacilli and 2.4% were Sarcina. Organisms isolated from plates incubated at 37 C were 72.9% streptococci, 12.9% micrococci, 8.9% microbacteria and 5.3% spore forming bacilli. Although several of the previously mentioned investigations indicated that adequate preheating of liquid milk and efficient drying temperatures eliminated the possibility of any pathogenic organisms surviving, there have been several outbreaks associated with staphylo- coccal food poisoning (2, 4, ll, 29, 37). A few food l6 poisoning outbreaks have also been associated with Sal- monellae and as a result several investigations were con— ducted with reference to the heat resistance of these organisms to spray drying and the effects of storage on their survival in nonfat dry milk. McDonough and Hargrove (42) concluded that although certain combinations of heat and moisture were sufficient in reducing the probability of Salmonellae survival during spray drying, these factors cannot be relied on for complete control. Adequate pasteurization destroyed Salmonellae in liquid milk, however, much higher temperatures were required to com- pletely destroy these organisms in concentrates. According to Julseth and Diebel (35) 15,000 to 30,000 Salmonellae per gram were sufficient to evoke a reaction in infants and adults, respectively. LiCari and Potter (38) reported that spray drying at commercial temperatures killed sub- stantial numbers of Salmonellae in skimmilk, but under no conditions did the treatment render the powder completely free of Salmonellae. These investigations clearly established the fact that pathogens and their toxins survived the spray drying operation, as evidenced by the number of outbreaks associ- ated with them. Therefore, low numbers of these organisms should be significant since these numbers will increase rapidly with hydration and incubation. 17 Changes in Microbial Content During Storagg According to Haines and Elliot (22) the rate of microbial die off in milk powder was influenced by moisture content, temperature and nature of the micro- organisms present. The amount of moisture in milk powder is directly related to its keeping quality because many microorganisms are incapable of product spoilage at low water content. As early as 1922 Supplee and Ashbaugh (51) observed that even if the microbial population of the powdered milk was significantly high, any relationship between bacterial numbers and keeping quality was eliminated by the lack of sufficient moisture to allow propagation. Later studies confirmed their observations. Foster et a1. (17) reported that low moisture levels prevented microbial metabolism, thereby eliminating microbial spoilage of dry milk. During the storage of roller dried milk the total microbial popu— lation decreased rapidly during the first month but became relatively constant after two to four months. A similar decrease was observed with spray dried milk during storage, however, the die-off was less marked. Spore formers and micrococci tended to survive longer than most other micro- organisms. Keogh (36) and Brockman (7) reported that micro- organisms which survived spray drying did not grow in the powder if the initial moisture level was low and if the 18 powder was protected from absorbing more moisture. Keogh suggested that microbial die-off during storage was attributed to the oxidation of enzymes. However, Brockman (7) suggested that the bacteriological quality or stability of the product which was stabilized by reduction in moisture content resulted from an interruption of processes necessary for microbial growth. Higginbottom (28) discussed the effect of relative humidity on bacterial numbers during storage and concluded that at relative humidities of 80-100% there was a rapid decrease in bacterial numbers followed by rapid growth of bacteria and molds. Relative humidities below 80% caused an increase in microbial population with maximum survival at approximately 10%. Data in Table 2 lists the moisture standards for milk powders. Decreases in microbial populations in milk powder stored at room temperature over prolonged storage were observed by Crossley and Johnson (12). In addition they observed that the rate of microbial reduction was acceler- ated by higher storage temperatures. McDonough and Hargrove (42) investigated the effect of moisture, temperature and length of storage on the survival of Salmonellae. The survival of these orga- nisms was directly influenced by temperature. No signifi- cant decrease in population was observed in powders stored 19 Table 2.-—Specific moisture standards for milk powders (1). Powder and grade Moisture content Spray Process Atmospheric Roller Not Greater Than Not Greater Than NFDM Extra 4.00% 4.00% Standard 5.00% 5.00% Dry Whole Milk Gas Packed Premium 2.25% Extra 2.50% Bulk Extra 2.50% Standard 3.00% Bulk Extra 3.00% Standard 4.00% Instant NFDM 4.50% at 26.6 C (79.8 F), however, as the temperature increased to 37.7 C (99.8 F), 43.3 C (109.9 F) and 50 C (122 F) the rate of destruction likewise increased. Although the higher storage temperature caused a decrease in microbial population, it proved to be undesirable due to the develop- ment of objectionable flavors. Survival of these organisms was also related to moisture content. A decrease in viable organisms occurred up to 15 to 20% moisture, above 20% destruction leveled off, and at 40% moisture, microbial growth was observed. LiCari and Potter (39) in a more recent study of microbial survival during drying and storage of nonfat dry 20 milk considered Salmonellae survival in powders incubated at 25 C (77 F) to 55 C (131 F). In four to eight weeks at 45 C (113 F) and 55 C there was a three or more log cycle reduction observed. However, at 55 C adverse flavors in addition to other physical defects were observed after one week storage. Storage at 25 C and 35 C (95 F) caused a much slower rate of destruction. In general, microbial die-off occurred at a dual rate with rapid destruction during the first two weeks of storage followed by rela- tively lower destruction rates. Bibek et al. (5) confirmed the work of LiCari and Potter. Their study showed that death rates, measured as total numbers, were quite high during the first two months of storage. Survival of these organisms in nonfat dry milk was considered to depend on five factors: a. Initial number of organisms present b. Strain of organism c. Temperature used during process d. Kind of product manufactured e. Conditions and duration of storage. During storage of the contaminated powders, different organisms exhibited different survival rates with some much more resistant to storage conditions than others. According to Crossley (10) decreases in the number of organisms in several powdered milk samples varied con- siderably between the powders. Some decreased 50% after 21 one month, in others reduction was relatively small even after six months. The author concluded that bacterial reduction depended primarily upon the nature of the flora. Spores survived for long periods, but streptococci died quite rapidly. By analysis of variance, Planine and Milohnoja (48) reported that the number of aerobic and facultative orga- nisms per gram of powder decreased parabolically during 12 weeks of storage. Fecal streptococci declined from 300 to 170 organisms/g, thermophiles declined from 92,000 to 31,000/g, thermodurics declined from 103,000 to 40,000/g and psychrophilic bacteria declined from 44,000 to 17,000/g. After four weeks of storage 34% of the samples met the desired microbial standards, after eight weeks 59.6%, and finally after twelve weeks 68% met the standards. Significance of Microbial Counts on Milk Powder Since utilization of nonfat dry milk as a food additive has increased over recent years, it is essential that it be wholesome and free from any objectionable bacteria. Even though it has been demonstrated that the number of organisms which survived spray drying decreased during subsequent storage, small numbers in milk powder proliferate rapidly upon hydration and incubation. Rela- tively low numbers of non-pathogenic or pathogenic 22 organisms introduced into a food product which was not heat treated or received an insufficient heat treatment prior to consumption can be significant, as evidenced by physical defects in the product or association with food poisoning outbreaks. Mattick et al. (45) suggested that plate counts on milk powder were related to plant cleanliness and sterility, preheating temperatures and bacteriological quality of the raw milk. Following this investigation Findlay et a1. (16) also observed that bacterial counts on spray dried powder were directly related to the preheating tempera- tures applied to raw milk and condensed milk. Counts were low when preheating temperatures of 190 F (87.7 C) and 200 F (93.3 C) were used. Counts were only slightly higher when 180 F (82.2 C) was used, but at 160 F (71.1 C) and 170 F (76.6 C) the counts were relatively higher. Von Loesecke (57) and Davis (14) both concluded that spray powders with low microbial populations were an indication of proper manufacturing practices, utilization of clean raw milk and proper storage following drying. Organisms which were present in the dry powder were those which survived forewarming and drying, or those intro- duced while packaging. Only the more resistant organisms were able to survive high temperatures during drum drying, whereas with spray drying, the less resistant forms sur- vived the process. Emphasis was placed on the theory that 23 the microbial content of dry milk furnished an index of product purity. Although Hammer and Babel (23) agreed that low bacterial counts indicated thorough heating during processing and adequate protective measures against contamination, they disagreed with the finding that the bacteriological quality of the raw milk influenced the bacteriological quality of the final powder. They deduced that plate counts on dry milk, whether high or low, could not be influenced by the microbiological quality of the original raw milk, since there was substantial microbial destruction during normal processing. Foster et a1. (17) and later Crossley (10) reported that the presence of coliforms in milk powder as in other pasteurized dairy products suggested unsanitary conditions and practices during production and storage. Isolation of molds from dry milk indicated excessive air contami- nation or poor handling during the process. McDivitt et a1. (41) and Hall and Hedrick (21) suggested that although the total bacterial count on dry milk may be low or the number of pathogens present is low or undetectable, this does not insure the safe quality of the product. Pathogenic staphylococci isolated from dry milks, even in low numbers, constitute a health hazard, especially if the product is held improperly after recon- stitution. The enterotoxin produced by these organisms 24 is not destroyed by the temperatures commonly used in pro- cessing spray dried milk. Control of the Microbial Con- tent of Dried Milk According to Keogh (36) the microbial content of milk powder was influenced more by the number of thermo— duric organisms in the raw milk than by the total bacterial content of the initial raw milk since those organisms were capable of surviving the temperatures used during pro- cessing. A review of the literature indicates that using high preheating temperatures has the most dramatic effect on controlling microbial populations of milk powder. However, there also must be good combinations of adequate plant and equipment sanitation, avoidance of air borne contamination, proper post processing and handling pro- cedures, selection of adequate storage containers and proper storage conditions. Galesloot and Stadhouders (19) suggested a few specific measures which could be taken in order to control the microbial content of dry milk. They suggested better control of conditions during milk production at the dairy farm in order to control the number of thermoduric orga- nisms which originated in the raw milk. Raising the pasteurization temperature was useful in controlling those thermoduric bacteria obtained from the dairy plant. 25 Bacterial counts may increase in the milk as it is moved from the pasteurizer to the drier. Consequently, adequate precautions should be taken to prevent the equipment involved during this process from becoming a continuous culture apparatus. Large balance tanks should not be used and capacities of the different sections of the installation should be in appropriate relationship to each other. The length of storage time should be short and the use of film evaporators in preference to circulation evaporators was suggested. Direct human contact with the milk during any part of the processing should be avoided. Concentrated milk should be pasteurized before it is pumped to the drier. This should control the thermoduric popu- lation in the powder. The final drying temperature was less effective for bacterial destruction than minimum pasteurization, therefore it had a limited effect on con- trolling the microbial content of the powder. Organisms which developed in the process before drying were mostly thermoduric organisms with a few non-thermoduric organisms such as E. aureus and Enterobacteriaceae. Normally these non-thermoduric organisms are destroyed during the drying operation. Finally, air-borne contamination during drying should be prevented. A drier which operates under nega- tive pressure utilizes outside air. Any bacteria which may be present in the outside air will not be heated to the same degree as the organisms which were present in 26 the concentrated milk, thus they have an increased chance of surviving. In order to prevent air borne contamination the authors suggested that air within the plant should not be used. Effect of Spray Drying on Various Microorganisms Crossley and Johnson (12) observed that high drying temperatures, 165 C (329 F) and above caused con- siderable bacterial destruction. The investigation stressed the significance of employing the highest pos- sible temperature without causing severe physical damage to the product in order to obtain relatively high thermal efficiency. Spray drying proved to be quite destructive to many bacteria. However, on the basis that some non- thermoduric organisms were able to survive drying, it was concluded that spray drying could not be relied on to eliminate pathogens. Crossley (10) observed that different organisms varied in their susceptibility to various spray drying conditions. He observed an increase in microbial survival when the inlet air temperature fell below 311 F (155 C) and a decrease in survival when the inlet air temperature was above 330 F (165.5 C). However, even when the inlet air temperature was raised to 410 F (210 C) some non-spore forming organisms survived spray drying. According to Jay (33) most microorganisms were destroyed during drying, however bacterial endospores 27 survived spray drying as did some yeasts, molds and some gram positive and negative bacteria. According to Hammer and Babel (23) spray drying caused a rapid loss of moisture during dehydration which kept the temperatures so low that it permitted survival of some of the more heat labile organisms. Generally the number of organisms which survived spray drying was rela- tively low. Those organisms were either believed to be protected in some manner that was not applicable to those organisms which were destroyed, or they were much more heat resistant. Foster et a1. (17) concluded that this pro- tective effect was caused by a layer of dried milk solids which remained around the bacterial cell, thus preventing complete dessication. Keogh (36) concluded that the pro- tein protected these heat sensitive organisms from the temperatures used during spray drying. LiCari and Potter (38) while working with Sal- monellae in nonfat dry milk suggested that the inlet air temperatures from 176 C (348.8 F) to 232 C (449.6 F) be used to spray dry nonfat dry milk. Exit air temperature dropped, in some instances, below 93 C (199.4 F). Product temperatures, however, were not maintained at this exit air temperature throughout drying due to cooling of the evaporated water. Consequently, during drying most orga- nisms were dehydrated below lethal temperatures and as soon as they were dried they became more resistant to the 28 temperatures that were used. In addition to being rela- tively resistant to those temperatures the organisms were assumed to be protected from heat by the milk solids. The microorganisms which survived spray drying were orga— nisms which entered the product via the evaporator, con- taminated air or a dirty drier. MATERIALS AND METHODS Selection and Propagation of MICroorganisms Stock cultures of E. SEAL! E. subtilis, and E. flavus were obtained from the Department of Food Science and Human Nutrition, Michigan State University. E. Egli was maintained in Nutrient Broth (Difco Laboratories, Detroit, MI), E. subtilis and E. flavus were maintained in Trypticase Soy Broth (BBL, Division of Becton, Dickin- son and Co., Cockeysville, MD). The stock cultures were stored at 4 C (39.2 F). Three days prior to the inocu- lation of E. ggll, E. subtilis or E. flavus into the con- centrated milk, one milliliter of the pure culture was transferred to 99 milliliters of Nutrient Broth or Trypticase Soy Broth. The inoculated broth was then incubated at controlled temperatures in a NBS Gyrotory Incubator Shaker (New Brunswick Scientific Co., Inc., New Brunswick, New Jersey). Broth cultures of E. flavus were held at 25 C and broth cultures of E. coli and E. subtilis were held at 35 C. (Agitation of these broth cultures caused a marked increase in microbial population over non- agitation.) 29 30 The active broth culture of the appropriate orga- nism was transferred every 24 hours for three days prior to inoculation of the milk. After the first 24 hour incubation period, the culture was diluted in phosphate buffer dilution blanks. The 24 hour cultures were plated according to the recommended Standard Plate count method in Standard Methods for the Examination 2: Dairy Products (3) with E. BELL on Violet Red Bile (VRB) Agar and E. flavus and E. subtilis on Plate Count Agar (PCA) (Difco) in order to determine the approximate population of each culture. Using these plating data as a basis, a suf- ficient amount of culture was inoculated into the milk to obtain the desired concentration of 1 x 106 organisms/ milliliter. Preparation of Concentrated Skimmilk Prior to each spray drying run, 50 gallons of raw whole milk were obtained from the Michigan State University Dairy Plant. The milk was separated, concentrated, and the skimmilk fraction was eventually subjected to a hydro- gen peroxide (H202)-catalase treatment (58) as an adjunct to pasteurization in order to substantially reduce the number of surviving bacteria in the pasteurized skimmilk. According to Hall and Hedrick (21) separation of cream can be accomplished with or without preheating the milk. Preheating milk to 68 to 86 F (20-30 C) enhances 31 efficiency of separation, but since there was a short holding period during processing, cold milk separation was more practical. The raw milk was separated into skimmilk and cream fractions using a DeLaval Air Tight Cream Separator (The DeLaval Separator Co., Poughkeepsie, NY). The skimmilk fraction was then pasteurized at 145 F (62.8 C) for 30 minutes. By utilizing a direct pipeline hook-up system, the pasteurized skimmilk was pumped from the vat and condensed in a vacuum evaporator (C. E. Rogers, Detroit, MI) to 35-40% total solids. During evaporation the percent total solids was determined by using a Baumé hydrometer (Fisher Scientific Company, Detroit, MI). Once the desired total solids was obtained the condensed skimmilk was drained from the vacuum pan into a sterilized ten gallon milk container. A chart converting Baumé readings to total solids was used to calculate the final percent total solids (21). Immediately following evaporation, the skimmilk fraction was cooled to 45-42 F (7.2-5.5 C) and placed in a walk-in cooler at 3.3 C (38 F) overnight. Eydrogen Peroxide-Catalase Treatment The germicidal characteristics of H202 have been known since its discovery by Thenard in 1818. In 1883, Schrodt established the use of H202 for preserving milk. 32 Since that time, numerous publications have been submitted relating to this subject. Luck (40) suggested that using H202 in treating milk served two purposes: a. Substitute short time treatment in place of pasteurization by heat, thus reducing the total bacterial count, and b. preservative to maintain the keeping quality for a longer period. He also suggested that treatment of milk with H202 caused a higher bacterial reduction than pasteurization by heat. According to Roundy (50) treating milk with H202 caused a selective destruction of many of the undesirable bacteria without adversely affecting the milk itself. The effective- ness of the H202 process depends on the temperature of the milk, the bacteriological quality of the milk at the time of sterilization, the concentration of H202 used, and the duration of the treatment. Luck (40), Roundy (50), and Walker and Harmon (58) found that adding 0.05% H202 to milk and heating to 120 F (48.9 C) established effective bactericidal conditions. In this investigation an 8.5-10 gallon sample of pasteurized, condensed skimmilk was heated in a steam- water jacketed kettle to 120 F (48.9 C) and treated with a 0.05% concentration of H202 (Mallinckrodt Chemical Works, St. Louis, M0) for a period of 20 to 30 minutes. 33 At the end of the 20 to 30 minute contact time, the milk was cooled to 100 F (37.7 C). It was essential that all of the H202 added to the milk be decomposed before adding the appropriate pure culture and this was accomplished by adding sterile catalase (Nutritional Biochemicals Corpor- ation, Cleveland, Ohio). Studies by Luck (40), Under- kofler (54), Walker and Harmon (58) show that cooling milk after the HZOZ-heat treatment enables the catalase to function more efficiently in breaking down H202 to water and oxygen. According to Roundy (50) adding excess catalase was not harmful. Four to five times the conceptual amount of catalase needed to destroy the H202, diluted with at least five times its volume of sterile water, was used. The milk was well agitated during the addition of H202 and catalase. In order to ensure that all of the residual H202 was decomposed by the catalase, a few drops of freshly prepared 25% solution of potassium iodide and 2% soluble starch solutions were added to two 5 ml samples of milk, one treated the other untreated. The resulting colors were compared; identical colors in the treated and untreated samples indicated complete destruction of H202 (50). In most instances the HZOZ-catalase treated sample showed a yellow discoloration, indicating that the residual H202 had not been decomposed. Based on this, the test was repeated at five minute intervals until no color change was noted in the treated sample. In the presence of 34 H202, a solution containing potassium iodide turns yellow due to the liberation of free iodine. At the end of the HZOZ-catalase sterilization treatment, a sample of the treated milk was plated on PCA and the plates incubated in order to evaluate the effectiveness of the treatment in reducing the number of bacteria which survived pasteurization. An investigation by Roundy (50) indicated that aerobic spore forming orga- the nisms are more resistant to destruction by H O 2 2’ coliform organisms are the least resistant and the sus- ceptibility of certain lactic acid organisms lies somewhere in between. According to Ito et a1. (32), aerobic spore formers, e.g., Bacillus subtilis globigii and Bacillus polynya were more resistant to H202 than anaerobic spore formers Clostridium sporogenes or Clostridium botulinum, with the exception of E. botulinum type B. Spggy Drying A vertical down-draft, direct gas fired, stainless steel spray dryer, manufactured by the Marriott Walker Corporation of Birmingham, Michigan was used. Three different exit air temperatures, 200 F, 180 F and 160 F (93.3, 82.2 and 71.1 C) were used to study the effects of drying temperatures on microbial destruction. On separate occasions a 1 x 106 concentration of the appropriate cul- ture was inoculated into the concentrated milk a few minutes before drying. The concentrated skimmilk had an 35 approximate temperature of 100 F (37.7 C). One third of the mixture was added and dried at each exit air tempera- ture, beginning with the higher temperature. The liquid sample was not added until equilibrium drying conditions were achieved at the desired outlet temperature. Samples were pumped by a high pressure pump to the top of the drier and atomized through a high pressure spraying nozzle into the chamber of the drier. A representative portion of each powder which had been dried at the specified temperature was collected asceptically in a large sterile glass jar. Since storage stability is an important factor in processing practice, the glass jars served as unique storage containers. The jars are impermeable to moisture vapor, consequently, they prevent absorption of moisture during storage. Absorption of moisture could very easily influence product deterior- ation due to chemical and bacterial changes (21). The following table gives the drying conditions used for each trial. Moisture Determinations The percent moisture in each dry milk sample was determined by the Karl Fischer titration, using a Beckman Model KF-2 Aquameter equipped with a duo-platinum electrode (Beckman Instruments, Inc., Scientific and Process Instru- ments Div., Fullerton, Cal.). The Karl Fischer reagent (Fisher Scientific Company, Detroit, MI) reacts 36 Table 3.--Drying conditions used to prepare skimmilk powder. Organism: E: flavus % Total solids - 35% % Total solids - 37% % Total solids - 40% Nozzle: Type SX Insert 65 Core 17A Homo pressure (psi) 1200 2300 2600 Pump speed 1.8 3.2 3.3 Air damper setting 12.7 12.7 12.7 Gas pressure 5.0 3.0 3.9 ASME nozzle 1.3 1.3 1.3 Inlet air, F 275 262 240 Exit air, F 200 180 160 Ambient air, F 78 78 78 Organism: E. subtilis Nozzle: Type SX Insert 65 Core 17A Homo pressure (psi) 1000 2100 2550 Pump speed 0.90 3.2 3.5- Air damper setting 13.4 13.4 13.4 Gas pressure 4.4 4.6 4.4 ASME nozzle 1.3 1.3 1.3 Inlet air, F 325 310 295 Exit air, F 200 180 160 Ambient air, F 75 75 75 Organism: E, coli Nozzle: Type SX Insert 65 Core 17A Homo pressure (psi) 1150 2300 3000 Pump speed 1.0 3.0 3.6 Air damper setting 13 13 13 Gas pressure 4.0 4.0 3.5 ASME nozzle 1.5 1.5 1.5 Inlet air, F 288 285 265 Exit air, F 200 180 160 Ambient air, F 75 75 75 37 quantitatively with the water in the sample to give a sharp chemical change (titration endpoint) that is detected electrochemically by the aquameter. Anhydrous methanol (Mallinckrodt) (active hydrogen compound) used to extract the moisture makes the entire reaction water specific, and in addition it acts as a stabilizer for the reaction. According to Joslyn (34) the following oxidation- reduction occurs in two steps: H (1) 12 + 802 + BQ—N + H20-———-)2©—-N< + I < >—n<:02 so H (2) Q—Ké 2 + CH3OH a ©—Nl80F 0—0 IGOF \A g “:0 o \o‘o—Oxo \U' 0 O\e=e-——_ _._8_, e 8 "c' .3. 5 — ~'5 a» o .J 4 I I I I I l Storage time (weeks) Fig. 3.--Survival of E. subtilis in spray dried skimmilk during storage at 25 C in milk dried with exit air temperatures of 160, 180 and 200 F. 47 Table 6.--Effect of storage on the number of E. coli in spray dried skimmilk powder. Storage time E. coli per gram of powder (weeks) Exit air temperature used at 25 C 200 F 180 F 160 F o 5.0 x 10% 5.1 x 103 1.1 x 10; l 3.1 x 102a 8.3 x 102 6.2 x 102 2 1.1 x 10la 3.7 x 101a 9.0 x 102 4 1.0 x 101a 8.0 x 10la 2.0 x 102a 8 1.0 x 10lb 1.0 x 10la 2.0 x 101a 12